In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a ∼480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-β) of the mouseKif1B-β gene in this region. The KIF1B-β gene had at least 47 exons, all of which had a classic exon–intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-β gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-β in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-β was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-β gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-β gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-β gene may not be a candidate for tumor suppressor gene of NB.
Neuroblastoma (NB) is derived from the neural crest and is the most common extracranial malignant solid tumor in childhood. Cytogenetic studies have suggested that deletion of the short arm of chromosome 1 (1p) occurs frequently in NB (Brodeur and Fong, 1989; Hayashi et al., 1989), and is associated with a poor prognosis (Hayasi et al., 1989; Brodeur et al., 1981; Caron et al., 1996). Molecular genetic studies have revealed several specific alterations in NB. One of these is amplification of the MYCN gene that is associated with a poor prognosis in NB (Seeger et al., 1985; Hayashi et al., 1989; Brodeur and Fong, 1989). Another is the loss of heterozygosity (LOH) at the 1p (Fong et al., 1989, 1992; Takita et al., 1995; Schwab et al., 1996), long arm of chromosome 11 (11q) (Srivatsan et al., 1993) and 14q (Srivatsan et al., 1993; Suzuki et al., 1989) in NB. In addition to those mentioned above, we recently found a LOH at 2q, 9p and 18q (Takita et al., 1995, 1998, 2000).
The commonly deleted region of chromosome 1 in NB maps to the 1p36 region between D1S244 and D1S243 (Fong et al., 1989, 1992; Schwab et al., 1996; Maris and Matthay, 1999). Several genes on distal 1p have been suggested as potential candidate tumor suppressor genes for NB. These include the cyclin dependent kinase (CDK) homologue CDC2L1 (Lahti et al., 1994); the zinc finger-containing transcription factors PAX7 (Shapiro et al., 1993), ID3 (Deed et al., 1994) and E2F2 (Saito et al., 1995); the tumor necrosis factor receptor 2 (TNFR2) (Beltinger et al., 1996); p73 gene (Kaghad et al., 1997; Yang et al., 2000) and DNA fragmentation factor 45 (DFF45) (Leek et al., 1997; Yang et al., 2001). None of them have shown any evidence as being a tumor suppressor gene for NB.
Homozygous deletions within the LOH region provide strong evidence that the genes or functions therein were lost and therefore suggests the presence of a tumor suppressor gene for NB. We searched for a homozygous deletion in NB cell lines by the polymerase chain reaction (PCR) using a high-density sequence tagged site (STS) content-map with a number of primer sets that mapped near D1S244 (Chen et al., 2001a). We observed a ∼480 kb homozygous deletion within chromosome band 1p36.2 in one of 24 NB cell lines, NB-1 (Chen et al., 2001b, in press). This region was flanked by B319M23, B203I23 and B223J10. We identified six known genes and a novel gene, the human KIF1B-β gene in this region. Here we performed genomic cloning, allelic imbalance (AI), and both expression and mutational analyses of the KIF1B-β gene in NB. We subsequently identified one missense mutation in the NB cell line and 38% of AI within the KIF1B-β gene in the fresh NB samples. RT–PCR analysis revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the gene was found in a subset of early- and advanced-stage NBs. These results suggested that the KIF1B-β gene might not be a candidate for the tumor suppressor gene in NB.
Homozygous deletion near D1S244
To identify the homozygous deletion of 1p36, we first selected 60 sets of STS and EST markers from the Genmap'99 in the NCBI Database (http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/maps) and the contig map of 1p36 made by our group (http://www.ncc.go.jp/research/1p-genome/) (Chen et al., 2001a). A panel containing DNA from 24 NB cell lines was used to perform PCR with 60 different sets of primers. All cell lines except for NB-1 revealed positive signals with all 60 sets of primers. The NB-1 cell line showed no signals at the regional just telomeric to D1S244. This region covered ∼480 Kb, which can be represented by the BAC clones B319M23, B203I23 and B223J10. Clone B203I23 was chosen for shotgun sequencing, because its insertion was the longest and it overlapped with both B319M23 and B223J10. One hundred (∼10%) colonies from the shotgun library were randomly picked for sequencing. All sequencing data were loaded on to the NCBI human EST database for a homology search. We found that these BAC clones contained the DFF45 gene, which has been mapped to 1p36.2–36.3 (Leek et al., 1997), the Cortistatin gene, a PGD homologue and an unknown gene that shared high homology to a major isoform of the murine brain Kif1B gene (Nakagawa et al., 1998, 1999; Conforti et al., 1999). We termed the human homologue of the murine brain major isoform of the Kif1B-β gene as KIF1B-β. A recent study revealed that there are six known genes (E4, SCYA5, PGD, Cortastatin, DFF45 and PEX14) in this region (Chen et al., 2001b, in press; Ohira et al., 2000).
Cloning cDNA and determining the genomic structure of the Kif1B gene
In order to search for EST homologues, sequences from the shotgun library for BAC B203I23 were cross-referenced to the NCBI Database. We found two sequences, part of which were 100% identical to a known EST KIAA0591, and either 5′ or 3′ of both these two sequences contained a classical exon–intron boundary structure. Therefore, we concluded that the gene encoding the KIAA0591 was present in BAC B203I23. The protein encoded by KIAA0591 shares high homology to the human axonal transporter of synaptic vesicles (H-ATSV), a homologue of the mouse Kif1A (Okada et al., 1995; Furlong et al., 1996). It shared a much higher homology to the C-terminal end of the murine brain major isoform of the Kif1B (KIF1B-β) (Nakagawa et al., 1998, 1999; Conforti et al., 1999), suggesting that KIAA0591 might be a part of the human homologue to murine KIF1B-β. Our prediction was confirmed by searching for a human EST homologue using the murine KIF1B-β. We did find one EST (AI95299) whose predicted amino acid sequence was about 96% identical to the murine Kif1B-β at an N-terminal 176 amino acids. Based on murine Kif1B-β there might be about a 1000 bp gap between the EST (AI95299) and KIAA0591. We designed a forward primer using the EST (AI95299) sequence and a reverse primer using the KIAA0591 sequence to perform RT–PCR using normal cDNA from peripheral lyphocytes. The sequence of the RT–PCR product was also homologous to the murine Kif1B-β. Thus, we cloned the full length of KIF1B-β (the GenBank accession number, AF257176). We confirmed that the KIF1B mRNA had at least two isoforms by Northern blotting using the 5′ end of KIF1B as a probe. The human KIF1B mRNA revealed a ∼12 Kb band (KIF1B-β) in an adult heart, brain, placenta, skeletal muscle, pancreas, spleen, thymus, prostate, testis, ovary, small intestine and colon, and in a fetal heart, lung, liver and brain (Figure 1). It also revealed an 8 kb band in adult testis (Figure 1). The KIF1B-β encoded for 1770 amino acids. Its N-terminal contained a conserved kinesin-like motor domain, and an ATP/GTP binding motif was located at amino acids 107–114 (Figure 2). Its C-terminus shared high homology to Kif1A and H-ATSV in the region that encoded a novel cargo domain. In KIF1B-β protein 94% of the amino acids are identical to the murine Kif1B-β (Figure 3). Analysis of the cDNA sequences for KIF1B-β, BAC B203I23, and B223J10 revealed that the KIF1B-β gene has at least 47 exons, all of which have a classic exon-intron boundary structure (Table 1).
Homozygous deletion of the KIF1B-β gene in the NB-1 cell line
Using PCR and Southern blotting we confirmed that the KIF1B-β gene was homozygously deleted in the NB-1 cell line (Figure 4). We did not find rearrangements or homozygous deletions of this gene in any other cell lines tested in this study.
Mutation and polymorphism of the KIF1B-β gene in NB
PCR–SSCP analysis of all the coding exons in the KIF1B-β gene (Table 2) revealed one missense mutation, silent mutations, and polymorphisms in the exons and introns (Table 3). We found that at codon 170, GAT (Asp) was changed to GAA (Glu) in six of the 23 (26%) NB cell lines and in seven of the 20 (35%) normal samples; at codon 1087, TAT (Tyr) was changed to TCT (Cys) in one of the 23 NB cell lines and one of the 50 normal samples, suggesting that they are polymorphisms. There were two silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT). There were also some changes in the KIF1B-β gene introns (Table 3). We found one missense mutation at codon 1554 with GTG (Val) change to ATG (Met) in the NB cell lines, but not in the 50 normal samples. This mutation was located at the cargo domain of KIF1B.
Expression of the KIF1B-β gene
RT–PCR analysis of the gene revealed obvious expression in all NB cell lines except NB-1, which showed no expression of the KIF1B-β gene. Reduced expression was found in two of eight early stage (stage I and II) NBs and three of eight advanced-stage (stage III and IV) NBs. There was no significant difference between the two groups.
AI of the KIF1B-β gene in NB
We chose two microsatellite markers centrimeric and telemetric to KIF1B-β for AI analysis. The incidence of AI at each locus was D1S244 (10/29; 35%), D1S1350 (8/25; 32%), L1 (3/8; 39%), and TT (4/11; 37%). AI for the KIF1B-β gene region (total of L1 and TT) was 6/16 (38%). Thus, the incidence of AI for the KIF1B-β gene was slightly higher than that for the D1S244 and D1S1350 loci Figure 5.
Abnormalities in Kif family proteins may affect the function of organelles and proteins that are necessary for cell differentiation and apoptosis, leading to carcinogenesis and other diseases. Mouse Kif1B-β is a microtubule-based putative anterograde motor protein for the axonal transport in neural cell. We cloned the human KIF1B-β gene from a region that was homozygously deleted in the NB cell line, NB-1. We physically mapped the KIF1B-β gene to 1p36.2, telomeric to D1S244 between WI-14873 and stSG 14169, within the commonly deleted region of NB (Schwab et al., 1996; Srivatsan et al., 1993; Maris and Matthay, 1999). Using two KIF1B-β polymorphic markers and two microsatellite markers in both sides of KIF1B-β, we found that the incidence of AI in the KIF1B-β gene was slightly higher than in the D1S244 and D1S1350 loci. Thus, the KIF1B-β gene may be considered as a candidate tumor suppressor gene for NB.
Using PCR–SSCP analysis, we found that at codon 170, the GAT (Asp) changed to GAA (Glu) in six of 24 (25%) NB patients. This change was considered to be a polymorphism, because we also found this change in 35% (7/20) of normal samples. We also found a rare polymorphism at codon 1087 (Tyr to Cys), because it was found in one of the 50 normal samples. There were two silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), respectively. There were also some changes in the KIF1B-β introns. We found one missense mutation at codon 1554 (Val to Met) (Table 3), which was located at the cargo domain of KIF1B-β, and is conserved in human and mouse. Although we do not know if it is functionally significant, a mutation of the KIF1B-β cargo domain could be important, because it may affect the cargo-binding specificity. Thus, in this PCR–SSCP study we found one missense mutation, but no nonsense mutations, suggesting that even though a homozygous deletion was found in one NB cell line (NB-1), the KIF1B-β gene may not be the real candidate for a suppressor gene in NB. However, we cannot rule out the possibility that the mutation resides in the promoter region of the KIF1B-β gene. Alternatively, the PCR–SSCP method may fail to detect the mutations because only around 90% of mutations can be detected by this method (Hayashi, 1991). Another explanation is that other tumor suppressor gene(s) for NB are present in the vicinity of this gene at 1p36.2. Previous RT–PCR–SSCP analysis of this gene except the motor domain did not show any mutations and polymorphisms in NB (Ohira et al., 2000), which is not compatible with our results. The cause of the discrepancy between the previous study and ours is unknown.
RT–PCR analysis of the KIF1B-β gene revealed that a reduced expression was found in two of eight early-stage NBs and three of eight advanced-stage NBs, respectively. These results are not compatible with a previous report (Ohira et al., 2000), which found that the KIF1B-β gene was expressed 3.6 times higher in favorable (early-stage) NBs than in unfavorable ones. The reason for the discrepancy between these two results is not yet known. Notably, there was obvious expression of the KIF1B-β gene in all NB cell lines except NB-1 in our study, suggesting that unfavorable NB also had considerable expression of this gene. Thus a further study is needed to clarify this point.
Kinesin was the first anterograde axonal transporter to be identified. It is a heterotetramer composed of two heavy chains and two light chains. The two heavy chains contain ATPase activity and bind to microtubules, while the light chains form a fan-like tail domain which is thought to be involved in binding to the organelle to be transported. A substantial number of other molecular motors similar to kinesin have been identified and classified as members of the kinesin superfamily (Kif) (Hirokawa, 1996, 1998). They differ in their molecular structure, relative position (N-terminal, C-terminal, or central) or the motor domain, and specificity for different cargoes (Hirokawa, 1996, 1998).
Kif1 and unc-104 are N-type proteins classified as first anterograde monomeric motors (Hirokawa, 1998). The Kif1 family has three members, Kif1A, Kif1B and Kif1C. Kif1A is a mouse homologue of the C. Elegans gene unc-104 and is an anterograde molecular motor responsible for the transport of synaptic vesicle precursors (Okada et al., 1995). Kif1A mutant mice died mostly within a day after birth showing motor and sensory defects (Yonekawa et al., 1998). However, there is no report of any mutation within the human homologue, H-ATSV, in human disease. Kif1B is the first cloned monomeric microtubule-based motor protein. It is a putative anterograde motor protein for the transport of mitochondria (Nangaku et al., 1994). Novel alternative transcripts of Kif1B that lack the mitochondria-binding domain were recently identified. This new isoform of Kif1B (Kif1B-α) contained a novel C-terminus, and is likely to have a different cargo-binding specificity (Nakagawa et al., 1998; Nakagawa and Hirokawa, 1999; Conforti et al., 1999; Gong et al., 1999). The Kif1B gene maps to the mouse distal chromosome 4 (Nakagawa et al., 1997; Conforti et al., 1999; Gong et al., 1999), in a region of synteny with the human chromosome band 1p36. The murine Kif1B gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 KDa kinesin, Kif1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, Kif1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1B gene (Conforti et al., 1999; Gong et al., 1999). Our Northern blotting revealed that human KIF1B had at least two isofrms. The long isoform (KIF1B-β) was expressed in a wide variety of different tissues, while the short isoform (KIF1B-α) was only detectable in adult testis. However, we were unable to clone the short isoform of KIF1B in our present study. It has been reported that a splicing form of Kif1B binds to the glucose transporter binding protein, thus providing a link between the glucose transporter and the cytoskeleton (Bunn et al., 1999). Further investigation for other isoforms of KIF1B may help us to understand the function of KIF1B. Kif1C is localized primarily at the Golgi apparatus, and is thought to be involved in transporting Golgi membrane (Dorner et al., 1998). The Kif1C can also form dimers and is associated with proteins of the 14-3-3 family (Dorner et al., 1999).
An axonal form of inherited peripheral neuropathy Charcot-Marie-Tooth (CMT2A), characterized by axonal loss, has been mapped to this location (1p36) in human (Saito et al., 1997). Inherited cutaneous melanoma and dysplastic nevus syndrome have also been mapped to this location (Greene, 1999). Analysis of the KIF1B gene in these diseases will clarify whether its mutations are causally linked to them.
In conclusion, we found a ∼480 kb homozygously deleted region within the commonly deleted region of NB in the NB-1 cell line. We determined the genomic structure of the KIF1B-β gene from this region, and found ∼40% of AI in this gene. However, finding only one missense mutation and an abundant expression of the KIF1B-β gene in various NB cell lines, suggests that the KIF1B-β gene may not be a candidate for the tumor suppressor gene of NB. A further functional study of this gene will be needed to clarify this result.
Materials and methods
Cell lines and primary tumors
Twenty-four NB cell lines were investigated. These included SCMS-N2∼SCMC-N5, NH-12, TGW, IMR-32, NB-1, NB-16, NB-19, NB-69, GOTO, SK-N-SH, CHP-134, LAN-1, LAN-2, and SJNB-1∼SJNB-5, SJNB-7, and SJNB-8. SCMC-N2∼SCMC-N5 were established by us (Komuro et al., 1993; Kong et al., 1997; Yang et al., 2000), while SJNB-1 ∼ SJNB-5, SJNB-7, and SJNB-8 were a generous gift from Dr AT Look. The other cell lines including NB-1 (Imashuku et al., 1973) were obtained from the Japanese Cancer Research Resources Bank (http://cellbank.nihs.go.jp). All cell lines were cultured in RPMI-1640 medium supplemented with 9% fetal bovine serum. Primary tumors were obtained from patients with NB at the time of initial surgery or biopsy, mainly in Saitama Children's Medical Center and the Affiliated Hospital of the University of Tokyo. Informed consent was obtained at each Institute.
Total RNA isolation and DNA extraction
Total RNA was extracted from the cell lines and tissues using the acid guanidine thiocyanate-phenol chloroform method (Komuro et al., 1993). High molecular weight DNA was extracted from 24 cell lines by proteinase K digestion and phenol/chloroform extraction (Kong et al., 1997; Yang et al., 2000).
Homozygous deletion screening with PCR
Sixty sets of STS or EST markers around D1S244 were obtained from the Genmap'99 or designed by ourselves based on the data from our 1p36 contig map (Chen et al., 2001a). The STS-content map is now accessible from our home page (http://www.ncc.go.jp/research/1p-genome/). Each NB cell line was examined with 60 PCRs using different primers sets. The reaction mixture was as follows: 50 ng of template DNA, 1 × PCR reaction Buffer, 2 μl of 2 mM dNTP, 20 pmol of each primer, and 1.25 u of Gold Taq Polymerase (Perkin-Elmer, New Jersey, USA) in a final volume of 25 μl. PCR was performed with a GeneAmp PCR system-9700 (Perkin Elmer, Norwalk, CT, USA): with the samples denatured at 94°C for 12 min followed by a 35 cycle amplification (94°C for 30 s, respective annealing temperature for 30 s, and 72°C for 30 s) and 7 min extention at 72°C. The PCR products were electrophoresed on a 3% agarose gel, and then stained with ethidiumbromide, before being photographed under UV.
Shotgun sequencing of BAC B203I23 and the database search
The BAC DNA from clone B203I23 was purified using a plasmid purification kit (Clontech, USA). The BAC DNA was used to make a shotgun library. About 10% of the colonies from the library were picked out for sequencing. The sequencing results were cross-referenced to the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/) for homology searching.
Cloning of the cDNA for KIF1B-β and determining its genomic structure
The NCBI Database and RT–PCR were used for cloning the full coding region of the KIF1B-β cDNA. The cDNA sequence of the KIF1B-β gene and BAC DNA from B203I23 and B372G17 were used to determine the genomic structure of the KIF1B-β gene.
PCR–SSCP and direct sequencing
Mutation analysis of the 47 exons of the KIF1B-β gene was carried out. The primers from introns used for this study are shown in Table 2. PCR amplification was performed with a GeneAmp PCR system-9700 (Perkin Elmer, Norwalk, CT, USA) as follows (Kong et al., 1997; Yang et al., 2000): denaturing at 94°C followed by a 35 cycle amplification (94°C for 30 s, appropriate annealing temperature for 30 s, 72°C for 30 s) and 7 min extention at 72°C. The reaction mixture contained 50 ng of genomic DNA or 1 μl cDNA, 0.8 μM of each primer, 100 μM of each dNTP, 1 × PCR buffer, and 0.25 units of Taq polymerase in a final volume of 5 μl. Following amplification, 45 μl of formamide denature dye mixture (95% formamide, 20 mM EDTA, 0.05% xylene cyanol and 0.05% bromophenol blue) was added to the PCR mixture, which was then denatured at 80°C for 5 min. A 2 μl aliquot of the denatured PCR product was applied to a non-denaturing polyacrylamide gel (TME buffer, pH 6.8) (Kukita et al., 1997) and electrophoresed at 20W for 3–4 h at room temperature. The gel was dried and exposed to X-ray film. After an abnormal band was identified, then direct sequencing was carried out on the PCR product of the band (Kong et al., 1997; Yang et al., 2000).
Total RNA from all 24 cell lines were available for RT–PCR. Sixteen primary tumor samples from NB patients were also available for RT–PCR. The 16 patients were grouped according to the classification of staging in NB (Evans et al., 1971). Of the 16 patients, eight were classified as early stage (five stage I and three stage II) and eight as advanced stage (two stage III and six stage IV). Randomly primed cDNA was made from total RNA, reverse transcribed with a cDNA synthesis Kit as previously described (Kong et al., 1997; Yang et al., 2000). The cDNA conversion mixture (1 μl) was amplified by PCR in a 10 μl reaction mixture. PCR conditions were 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C for 28 cycles, followed by 10 min at 72°C. The following primer set was used for RT–PCR of the KIF1B-β gene : PS (exond 43, 44), 5′-CAGTGACTGTAAGTTGTCTGATATA-3′ and PA (exon 46), 5′-GTAAAGAGGCTCCTTGAAAT-3′. PCR products were electrophoresed on a 2.0% agarose gel and stained with ethidiumbromide.
Ten micrograms of DNA were digested with BamHI, and electrophoresed on 1.0% agarose gels. The samples were then transferred to charged nylon filters, before being hybridized, and exposed to X-ray film (Kong et al., 1997; Yang et al., 2000). The KIF1B-β cDNA was used as a probe.
Fifty matched samples from NB patients (tumor and normal peripheral blood) were used for the AI study (Takita et al., 1995, 1998, 2000). Two polymorphic markers (L1 and TT) selected from the KIF1B-β gene, and two microsatallite markers, D1S244 and D1S1350, were used for a AI study on the matched samples from 50 patients. An AI detected with each set of primers was considered to have occurred if the reduction rates in the signal intensities of the tumors were more than 40% (Takita et al., 1998, 2000).
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We thank Mrs Soma and Mrs Soga for their excellent technical assistance. We express our appreciation to Professor Thomas A Look, Harvard Medical School, Dana-Farber Cancer Institute, for his generous gift of the NB cell lines. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan and a Grant-in-Aid for Scientific Research on Priority Areas and Grant-in-Aid for Scientific Research (B) and (C) from the Ministry of Education, Science, Sports and Culture of Japan.
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